WO2011054995A2

WO2011054995A2 - PROPHYLACTIC FLU VACCINES FROM VIRAL CAPSIDS OF BIRNAVIRUS CONTAINING THE M2e ANTIGEN OF THE FLU VIRUS

Info

Publication number: WO2011054995A2
Application number: PCT/ES2010/070716
Authority: WO
Inventors: Thomas Zurcher; Cayetano Von Kobbe; Juan José BERNAL; Ignacio JIMÉNEZ TORRES; María VELA CUENCA; Ana Diaz Blazquez; Miguel Angel Llamas Matias; Diana Martin Lorenzo; Arcadio GARCÍA DE CASTRO
Original assignee: Chimera Pharma, S. L. U.
Priority date: 2009-11-06
Filing date: 2010-11-05
Publication date: 2011-05-12
Also published as: WO2011054995A3

Abstract

The invention relates to prophylactic vaccines for preventing flu in humans. The vaccines according to the invention are especially formed by chimeric pseudo-viral capsids of birnavirus containing the M2e antigen of the flu virus.

Description

FLU PROPHACACTIC VACCINES FROM VIRAL CAPSIDES OF BIRNAVIRUS CONTAINING THE M2e ANTIGEN OF THE VIRUS OF THE FLU. The invention relates to prophylactic vaccines for the prevention of influenza in humans. In particular, the vaccines of the present invention are formed by birnavirus chimeric pseudo-viral capsids containing the influenza virus M2e antigen. STATE OF THE PREVIOUS TECHNIQUE

The flu represents a major global health problem. It is estimated that each year the flu affects 1 billion people, of which between 3 and 5 million develop a severe form of the disease that causes between 300,000 and 500,000 deaths.

Every year around 200 million people in the world are vaccinated with prophylactic vaccines against seasonal flu that may contain more than one strain of inactivated Influenzavirus. The need to obtain a new seasonal vaccine every year is due to the great variability of the Neuraminidase (NA) and Hemagglutinin (HA) antigens of Influenzavirus surface proteins that cause influenza. However, the M2 surface protein ectodomain (in English, M2e Matrix Protein 2 Ectodomain) is an antigen that is highly conserved among different strains of influenza viruses of human origin and has been proposed as a candidate for "universal" vaccines that would confer protection against different strains of seasonal influenza [Schotseart M. et al. Expert Rev. Vaccine (2009) 8 (4): 499-508].

There are currently clinical developments of universal vaccines that incorporate the M2e antigen. Among them, it is worth mentioning those in which the M2e antigen is bound or fused to other biological entities, such as the TLR5 flagelin ligand [Huleatt W et al. Vaccine (2008) 26, 201— 214], the hemocyanin of Megathura Crenulata (KLH) or the outer membrane protein complex of Neisseria meningitidis (OMPC, Outer Membrane Protein Complex) [Fan J et al. Vaccine (2004) 22: 2993-3003].

An example of the presentation of antigens on biological macrostructures is chimeric pseudo-viral particles (VLP) formed from structural viral proteins that incorporate antigens relevant to a disease. In this sense there are precedents that incorporate the M2e antigen in Hepatitis B VLP [WO9907839; Neirynck S et al Nat. Med (1999) 5 (10): 1 157-1163; From Filíte M et al. (2006) Vaccine 24 (44-46): 6597-6601; From Filíte M et al. (2008) Vaccine 26 (51): 6503-6507]. Among the VLPs proposed for the presentation of different antigens are those derived from the Infectious Bursitis Virus (IBDV). IBDV belongs to the Birnaviridae family and is the causative agent of Gumboro disease in birds. IBDV viral particles are icosahedral with symmetry T = 13 and are formed by 260 trimers of the VP2 protein (37 kDa) reinforced internally by the structural protein VP3 (29 kDa). In the virion morphogenesis process the protein components of the viral capsid result from the proteolysis of the precursor polypeptide pVP2-VP4-VP3 (109 kDa) to release the VP2 precursor of 512 amino acids (pVP2 ₅₁₂ ), VP4 and VP3. Subsequent amino acid cleavage at the carboxyl terminus of pVP2 ₅₁₂ results in the mature form of VP2 441 amino acids in length (VP2 ₄₄ i) present in the virion [Da Costa B. et al. J. Virology (2002) 76 (5): 2393-2402]. VP2 of different strains of IBDV have a protein sequence homology of more than 80%. The VP2 of other Birnaviridae share with IBDV homologies in their protein sequence of 40% in the case of aquatic Birnavirus and 30% of Drosophila melanogaster Birnavirus [Coulibaly F. et al. (2005) Cell 25,120 (6): 761-772]. Expression of the precursor polypeptide pVP2-VP4-VP3 of IBDV in eukaryotic cells results in the formation of icosahedral VLPs of T = 13 symmetry, identical to the IBDV native capsids [Martínez-Torrecuadrada JL. et al. (2001) J. Virology 75 (22): 10815-10828]. Likewise, the expression of VP2 in eukaryotic cells in the absence of other IBDV proteins causes the formation of VLP icosahedral particles of T = 1 symmetry smaller than the IBDV virions [Martinez-Torrecuadrada JL. et al. (2003) Vaccine 21 (17-18): 1952-1960]. This feature has been exploited for the design and expression of chimeric VLPs that incorporate the BT antigen from the FMD virus in VLP T = 1 from fusions of sequences encoding said antigen at the carboxyl end of the gene of the VP2 protein [WO2007009673].

The ability of VP2 containing fusions or insertions of different nature to form IBDV VLPs of type T = 1 efficiently in an eukaryotic expression system depends, to a large extent, on the length of the VP2 [WO2005105834; Saugar I. et al. (2005) Structure 13 (7): 1007-1 1 17], of the place of insertion of these inserts in the VP2 sequence, and of the sequence of the inserts introduced in VP2 [Rémond M. et al (2009) Vaccine 27 {1): 93-8]. However, the ability of the chimeric VLPs obtained to induce an adequate immune response in an animal model is unpredictable.

DESCRIPTION OF THE INVENTION

In the design of universal prophylactic influenza virus vaccines, it would be desirable to have an M2e antigen presentation system that generates a specific and effective immune response. In the present invention sequences coding for VP2 of IBDV and M2e of influenza virus are combined in order to obtain an effective vaccine in the prevention of influenza. It is not obvious which sequences of M2e or VP2 may be suitable in the generation of the vaccine, nor the optimal fusion or insertion site of M2e with VP2. For this reason, a chimeric VLP T = 1 search process is carried out in the present invention from fusions and insertions of sequences encoding M2e in the IBDV VP2 gene that result in the selection of the most effective chimeric VLPs in the prevention of influenza caused by Influenzavirus.

The insertion of non-birnavirus sequences in the VP2 gene results in modifications in the three-dimensional structure of the encoded protein that negatively affect its ability to self-assemble and form pseudo-viral capsids efficiently. This negative effect depends not only on the insertion site, but also on the amino acid sequence and length of the insert. Therefore, it is not obvious a priori which insertion points and inserted amino acid sequences result in an efficient formation of chimeric VLPs. Likewise, the formation of VLP incorporating sequences containing M2e is not a sufficient condition to generate an efficient prophylactic vaccine against the influenza virus, but this depends on the final disposition of the antigens containing M2e in the resulting chimeric VLP. Moreover, it is known that the immune system responds more potently against antigen repeats and, therefore, the particles selected in the present invention incorporate two or three copies of M2e, preferably two. The chimeric VLPs of the present invention are obtained from a selection process in which the optimal arrangement of the M2e sequences for VLP formation and the VP2 insertion sites that give rise to chimeric VLPs with a greater efficacy in prophylaxis against influenza virus.

As described, without limitation, in Example 1, the present invention provides pseudo-viral particles originating from insertions of M2e sequences encoding the influenza virus into the gene encoding Birnavirus VP2 and effective in prevention. of the flu caused by different variants of the virus that causes the disease. A first aspect of the invention relates to a chimeric pseudo-viral (VLP) particle (hereinafter, chimeric VLP of the invention) formed by a fusion protein (hereinafter, fusion protein of the invention) which understands:

- a subunit (a) consisting of the Birnavirus pVP2 protein or a fragment thereof, and

- a subunit (b) comprising an influenza virus M2e antigen,

where the subunit (b) is inserted in the subunit (a).

The term "pseudo-viral capsid," "pseudo-viral particle" or "VLP" (in English Virus-Like Partióle) refers to a three-dimensional nanometric structure formed by the assembly of structural viral proteins. In the present invention, the structural viral proteins that form the pseudo-viral particle of the invention are fusion proteins comprising the pVP2 protein of a Birnavirus or a fragment thereof and at least one M2e antigen of the Influenza virus (Influenzavirus ). The term Birnavirus refers to any virus of the family Birnaviridae, belonging to Group I I I according to the Baltimore Classification. The Birnaviridae family consists of the genera Avibirnavirus, Aquabirnavirus, Blosnavirus and Entomobirnavirus. Preferably, the Birnavirus is from the Avibirnavirus family, and more preferably, the infectious Bursitis Virus (IBDV).

The term "infectious bursitis virus" or "IBDV" (IBDV) refers to viruses of the family Birnaviridae and genus Avibirnavirus causing Gumboro disease in chickens and belonging to Group II I of the Baltimore Classification. Preferably, the IBDV is the IBDV strain Soroa. The Birnavirus genome consists of two linear double-stranded RNA molecules called A and B, which encode 5 proteins. The VP2 gene, embedded in segment A, encodes the VP2 protein precursor protein (pVP2). The elimination of sequences from the carboxyl terminus of pVP2 by proteolysis results in the mature VP2 protein, which is the main protein that constitutes the viral capsid.

The term "pVP2 protein" or "pVP2", as used herein, refers to the VP2 precursor protein encoded by the VP2 gene of a Birnavirus. Preferably, this term refers to the 512 amino acid VP2 precursor protein (VP2 ₅ and ₂ ) of IBDV.

The amino acid sequence of the VP2 ₅ 12 protein of the IBDV strain Soroa (SEQ ID NO: 1) is deposited with the accession number AAD30136 in NCBI (from the National Center for Biotechnology Information). VP2 ₅₁₂ protein from other strains of IBDV has at least 80% identity with SEQ ID NO: 1. Therefore, in a preferred embodiment, the term pVP2 refers to a protein with at least 80%, 85%, 90%, 95%, 98% or 99% identity, with SEQ ID NO: one . In a more preferred embodiment the term pVP2 refers to SEQ ID NO: 1.

The term "identity", as used in this description, refers to the proportion of identical amino acids between two amino acid sequences that are compared. The percentage of identity existing between two amino acid sequences can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences.

The term "fragment", as used herein, refers to a portion of the pVP2 protein, of at least 400 amino acids, capable of forming VLP. This term includes, therefore, the mature VP1 protein of 441 amino acids of the IBDV (VP2 ₄₄ i). In a preferred embodiment of this first aspect of the invention, the subunit (a) of the fusion protein that forms the chimeric VLP of the invention consists of a protein with at least 80% identity with SEQ ID NO: 1 or A fragment of it. In a more preferred embodiment, the subunit (a) of the fusion protein that forms the chimeric VLP of the invention consists of a protein with SEQ ID NO: 1 or a fragment thereof. In an even more preferred embodiment, the subunit (a) of the fusion protein that forms the chimeric VLP of the invention consists of SEQ ID NO: 2.

In a preferred embodiment, the subunit (b) is inserted into the P region of the subunit (a). The term "P region", as used herein, refers to the four external domains (BC, DE, FG and H 1) of the pVP2 protein or the amino acid sequences corresponding to said domains in a pVP2 protein fragment.

The four external domains of the IBVV strain Soroa strain pVP2 protein correspond to the sequences between amino acids Q219-G224 (BC domain), R249-L255 (DE domain), T283-D287 (FG domain) and S315-Q324 (domain Hl). In parentheses are the first and last amino acid of each domain and its corresponding position in the pVP2 sequence of IBDV.

The term "BC domain of the P region" therefore refers to the amino acid sequence between the amino acid of position 219 and the amino acid of position 224 of the pVP2 protein. Preferably, this term refers to the amino acid sequence between Q219 and G224 of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between the Q219 and G ₂ 24 SEQ ID NO: 2. The term "DE domain of the P region" thus refers to the amino acid sequence between the amino acid of position 249 and the amino acid of position 255 of the pVP2 protein. Preferably, this term refers to the amino acid sequence between R ₂₄ g and l ₂₅ of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between R ₂₄ g and L ₂₅ 5 of SEQ ID NO: 2.

The term "FG domain of the P region" thus refers to the amino acid sequence between the amino acid of position 283 and the amino acid of position 287 of the pVP2 protein. Preferably, this term refers to the amino acid sequence between T ₂₈ 3 and D ₂₈ 7 of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between T ₂₈ 3 and D ₂₈ 7 of SEQ ID NO: 2.

The term "Hl domain of the P region" thus refers to the amino acid sequence between the amino acid of position 315 and the amino acid of position 324 of the pVP2 protein. Preferably, this term refers to the amino acid sequence between S315 and Q3 ₂₄ of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between S315 and Q324 of SEQ ID NO: 2. In a preferred embodiment of this first aspect of the invention, the subunit (b) is inserted into the P region of SEQ ID NO: 2.

In a preferred embodiment of this first aspect of the invention, the subunit (b) of the fusion protein that forms the VLP of the invention is inserted into the Hl domain of the P region pVP2 or a fragment of pVP2. In a more preferred embodiment, the subunit (b) of the fusion protein that forms the VLP of the invention, is inserted between amino acids D ₃₂ 3 and Q324 of pVP2 or a fragment of pVP2.

In a more preferred embodiment of this first aspect of the invention, the subunit (b) of the fusion protein that forms the VLP of the invention is inserted in the Hl domain of the P region of SEQ ID NO: 2. In A more preferred embodiment, the subunit (b) of the fusion protein that forms the VLP of the invention, is inserted between amino acids D ₃₂₃ and Q ₃₂₄ of SEQ ID NO: 2.

The term "M2e antigen" or "M2e" (Matrix Protein 2 Ectodomain) refers to the flu virus M2 matrix protein antigen that contains the extracellular region of that protein. The term M2e antigen includes antigens of the influenza virus M2 matrix protein, such as, but not limited to, the human, swine or avian influenza virus. In a preferred embodiment, the term "M2e antigen" refers to the antigen of the human influenza virus M2 matrix protein that contains the extracellular region of said protein, whose sequence is SEQ ID NO: 3. Therefore, the term M2e, preferably, it refers to a polypeptide with at least 60%, 70%, 80%, 90%, 95% or 99% identity, with SEQ ID NO: 3. More preferably, the term is refers to a polypeptide with the amino acid sequence SEQ ID NO: 3.

The replacement of the tanks with serines in SEQ ID NO: 3 confers greater stability and / or prevents aggregation of the M2e antigen. Therefore, in a preferred embodiment, the term M2e antigen refers to the influenza virus M2 matrix protein antigen containing the extracellular region of said protein in which some or all tanks have been replaced by serines. In a more preferred embodiment, this term refers to a polypeptide with the amino acid sequence SEQ ID NO: 4. In a preferred embodiment of this first aspect of the invention, the subunit (b) of the fusion protein comprises two M2e antigens linked by a hydrophilic polypeptide. Subunit (b) is inserted into subunit (a) to give rise to the fusion protein that forms the chimeric VLP of the invention. The term "inserted" means that the amino acid sequence of the subunit (a) is divided into two parts (a1) and (a2), among which is the amino acid sequence of the subunit (b). The union between the amino acid sequence of the subunit (b) and the amino acid sequence of each of the subunits (a1) or (a2) can be direct or by one or two spacer polypeptides.

The term "spacer polypeptide" or "linker", as used herein, refers to a short amino acid sequence, preferably, up to 15 amino acids in length, more preferably, up to 10 amino acids in length, even more preferably, up to 5 amino acids in length, located either between the amino acid sequence of the subunit (b) and the amino acid sequence of the subunit (a), or between the M2e antigens that are part of the subunit (b ).

In a preferred embodiment, the spacer polypeptide (p) is a hydrophilic polypeptide. The term "hydrophilic spacer polypeptide" or "hydrophilic linker" are sequences comprising between 2 and 8 hydrophilic amino acids (h), such as histidine (H), glutamine (Q), asparagine (N), lysine (K), aspartic acid (D), glutamic acid (E), arginine (R), serine (S) and glycine (G). In a more preferred embodiment, the hydrophilic polypeptide is a polypeptide consisting of 4 hydrophilic amino acids (hhhh). When the union between the subunits (a) and (b) of the fusion protein that forms the VLP of the invention is direct, the amino acid of the carboxy-terminal end of the part (a1) of the subunit (a) forms a bond peptide with the amino acid of the amino-terminal end of the subunit (b) and the amino acid of the carboxyl-terminal end of the subunit (b) forms a peptide bond with the amino acid of the amino-terminal end of part (a2) of the subunit (a) , as represented in the following scheme:

Nt- (a1) -Ct ~ Nt- (b) -Ct ~ Nt- (a2) -Ct where (a1) represents a part of the subunit (a), (a2) represents the other part of the subunit (a) , (b) represents the subunit (b), Nt represents the amino-terminal end of the corresponding subunit, Ct represents the carboxyl-terminal end of the corresponding subunit, and ~ represents a peptide bond between the different units of the protein of fusion of the invention. When the union between the subunits (a) and (b) of the fusion protein that forms the VLP of the invention is carried out by means of two spacer polypeptides, which may be the same or different, the amino acid of the carboxyl-terminal end of the part ( a1) of the subunit (a) forms a peptide bond with the amino acid of the amino-terminal end of a first spacer polypeptide (p1), the amino acid of the carboxyl-terminal end of this first spacer polypeptide (p1) forms a bond with the amino acid of the amino-terminal end of the subunit (b), the amino acid of the carboxyl-terminal end of the subunit (b) forms a peptide bond with the amino acid of the amino-terminal end of a second spacer polypeptide (p2) and the amino acid of the end The carboxyl-terminal of this second spacer polypeptide (p2) forms a bond with the amino acid of the amino-terminal end of part (a2) of the subunit (a), as depicted in the following scheme:

Nt- (a1) -Ct ~ Nt- (p1) -Ct ~ Nt- (b) -Ct ~ Nt- (p2) -Ct ~ Nt- (a2) -Ct where (a1) represents a part of the subunit (a), (a2) represents the other part of the subunit (a), (b) represents the subunit (b), (p1) represents a first spacer polypeptide, (p2 ) represents a second spacer polypeptide, Nt represents the amino-terminal end of the subunit or the corresponding spacer polypeptide, Ct represents the carboxyl-terminal end of the subunit or the corresponding spacer polypeptide, and ~ represents a peptide bond between the different units of the fusion protein of the invention. When the union between the subunits (a) and (b) of the fusion protein that forms the VLP of the invention is carried out by means of only one spacer polypeptide, the subunit (b) at one end is joined with one of the parts of the subunit (a) directly by peptide bonding and at the other end is linked to the other part of the subunit (a) by a spacer polypeptide, as represented in the following schemes:

Nt- (a1) -Ct ~ Nt- (b) -Ct ~ Nt- (p) -Ct ~ Nt- (a2) -Ct Nt- (a1) -Ct ~ Nt- (p) -Ct ~ Nt- ( b) -Ct ~ Nt- (a2) -Ct where (a1) represents a part of the subunit (a), (a2) represents the other part of the subunit (a), (b) represents the subunit (b) , (p) represents a spacer polypeptide, Nt represents the amino-terminal end of the subunit or the corresponding spacer polypeptide, Ct represents the carboxyl-terminal end of the subunit or the corresponding spacer polypeptide, and ~ represents a peptide bond between the different fusion protein units of the invention.

In a preferred embodiment, the fusion protein that forms the chimeric VLP of the invention comprises, in addition to subunit (a) and subunit (b), one or two hydrophilic spacer polypeptides between the amino acid sequence of subunit (b) and the amino acid sequences of the subunit (a). In a more preferred embodiment, the fusion protein that forms the chimeric VLP of the invention comprises, in addition to the subunit (a) and the subunit (b), two hydrophilic spacer polypeptides between the amino acid sequence of the subunit (b) and the amino acid sequences of the subunit (a). In a more preferred embodiment, the subunit (b) of the fusion protein that forms the chimeric VLP of the invention comprises two M2e antigens (M2e-1 and M2e-2), which may be the same or different from each other. The union between the amino acid sequence between these two antigens M2e-1 and M2e-2 of the subunit (b) can be direct or by a spacer polypeptide.

When the binding between the M2e-1 and M2e-2 antigens of the subunit (b) of the fusion protein that forms the VLP of the invention is direct, the carboxyl-terminal amino acid of a first M2e-1 antigen forms a peptide bond with the amino acid of the amino-terminal end of a second M2e-1 antigen, as depicted in the following scheme:

~ Nt- (M2e-1) -Ct ~ Nt- (M2e-2) -Ct ~ where (M2e-1) represents one of the M2e antigens, (M2e-2) represents the other M2e antigens, Nt represents the amino end -terminal of the corresponding antigen, Ct represents the carboxyl-terminal end of the corresponding antigen, and ~ represents a peptide bond between the different units of the subunit (b) of the fusion protein of the invention. When the binding between the M2e-1 and M2e-2 antigens of the subunit (b) of the fusion protein that forms the VLP of the invention is performed by a spacer polypeptide, the amino acid of the carboxyl-terminal end of a first M2e antigen -1 forms a peptide bond with the amino-terminus amino acid of a spacer polypeptide (p) and the amino acid of the carboxy-terminal end of the spacer polypeptide (p) forms a peptide bond with the amino acid of the amino-terminal end of a second M2e-2 antigen, as represented in the following scheme: ~ Nt- (M2e-1) -Ct ~ Nt- (p) -Ct ~ Nt- (M2e-2) -Ct ~ where (M2e-1) represents one of the M2e antigens, (M2e-2) represents the other M2e antigens, (p) represents a spacer polypeptide, Nt represents the amino-terminal end of the corresponding antigen or spacer polypeptide, Ct represents the carboxyl-terminal end of the corresponding antigen or spacer polypeptide, and ~ represents a peptide bond between the different units of the subunit (b) of the fusion protein of the invention.

In a more preferred embodiment, the subunit (b) of the fusion protein that forms the chimeric VLP of the invention comprises three M2e antigens (M2e-1, M2e-2 and M2e-3), which may be the same or different from each other. . The union between the amino acid sequence between these different antigens M2e-1, M2e-2 and Me-3 of subunit (b) can be direct or by one or two spacer polypeptides.

When the binding between the M2e-1, M2e-2 and M2e-3 antigens of the subunit (b) of the fusion protein that forms the VLP of the invention is carried out by two spacer polypeptides, which may be the same or different from each other. , the amino acid of the carboxyl-terminal end of a first M2e-1 antigen forms a peptide bond forms a peptide bond with the amino acid of the amino-terminal end of a first spacer polypeptide (p1), the amino acid of the carboxyl-terminal end of this first Spacer polypeptide (p1) forms a bond with the amino acid of the amino-terminal end of a second M2e-1 antigen, the amino acid of the carboxyl-terminal end of this second M2e-2 antigen forms a peptide bond with the amino acid of the amino-terminal end of a second spacer polypeptide (p2) and the carboxyl-terminal amino acid of this second spacer polypeptide (p2) forms a bond with the amino acid of the amino-terminal end of a te rcer M2e-3 antigen, as depicted in the following scheme: ~ Nt- (M2e-1) -Ct ~ Nt- (p1) -Ct ~ Nt- (M2e-2) -Ct ~ Nt- (p2) -Ct ~ Nt- (M2e-3) -Ct ~ where (M2e -1) represents a first M2e antigen, (M2e-2) represents a second M2e antigen, (M2e-3) represents a third M2e antigen, (p1) represents a first spacer polypeptide, (p2) represents a second spacer polypeptide, Nt represents the amino-terminal end of the corresponding antigen or spacer polypeptide, Ct represents the carboxyl-terminal end of the corresponding antigen or spacer polypeptide, and ~ represents a peptide bond between the different units of the subunit (b) of the fusion protein of the invention.

When the binding between the M2e-1, M2e-2 and M2e-3 antigens of the subunit (b) of the fusion protein that forms the VLP is performed by only a spacer polypeptide, one of the junctions between the M2e antigens is a direct binding by peptide bond, while another of the junctions takes place by means of a spacer polypeptide, as represented in the following schemes:

~ Nt- (M2e-1) -Ct ~ Nt- (p) -Ct ~ Nt- (M2e-2) -Ct ~ Nt- (M2e-3) -Ct ~

~ Nt- (M2e-1) -Ct ~ Nt- (M2e-2) -Ct ~ Nt- (p) -Ct ~ Nt- (M2e-3) -Ct ~ where (M2e-1) represents a first M2e antigen , (M2e-2) represents a second M2e antigen, (M2e-3) represents a third M2e antigen, (p) represents a spacer polypeptide, Nt represents the amino-terminal end of the corresponding antigen or spacer polypeptide, Ct represents the end carboxyl-terminal of the corresponding antigen or spacer polypeptide, and ~ represents a peptide bond between the different units of the fusion protein of the invention. In a preferred embodiment, the subunit (b) of the fusion protein that forms the chimeric VLP of the invention comprises two M2e antigens, linked together in addition to the subunit (a) and subunit (b), one or two polypeptides hydrophilic spacers between the amino acid sequence of the subunit (b) and the amino acid sequences of the subunit (a).

In a more preferred embodiment, the fusion protein where the subunit (b) comprises two M2e antigens, linked together by a hydrophilic polypeptide, wherein said subunit (b) is inserted into the subunit (a) by two hydrophilic spacer polypeptides, such and as represented in the following scheme: ~ Nt- (a1) -Ct ~ Nt- (p1) -Ct ~ Nt- (M2e-1) -Ct ~ Nt- (p2) - Ct ~ Nt- (M2e-2 ) -Ct ~ Nt- (p3) -Ct ~ Nt- (a2) -Ct ~, where (M2e-1) represents a first M2e antigen, (M2e-2) represents a second M2e antigen, (p1) represents a first spacer polypeptide, (p2) represents a second spacer polypeptide, (p3) represents a third spacer polypeptide, Nt represents the amino-terminal end of the antigen or the corresponding spacer polypeptide, Ct represents the carboxyl-terminal end of the antigen or the corresponding spacer polypeptide , and ~ represents a peptide bond between the different units of the fusion protein of the inv portion.

In a preferred embodiment, the amino acid sequence of ~ Nt- (p) -Ct ~ Nt- (M2e-1) -Ct ~ Nt- (p) -Ct ~ Nt- (M2e-2) -Ct ~ Nt- ( p) -Ct ~ according to the previous scheme is SEQ ID NO: 7.

A preferred embodiment of this first aspect of the invention relates to a chimeric VLP formed by a fusion protein comprising SEQ ID NO: 7 inserted between amino acids D323 and Q324 of SEQ ID NO: 2. A more preferred embodiment, refers to a chimeric VLP formed by a fusion protein whose amino acid sequence is SEQ ID NO: 8.

A second aspect of the invention relates to a process for obtaining the chimeric VLPs of the invention, which comprises culturing a host cell comprising a nucleic acid encoding the fusion protein of the invention, under conditions that allow expression. from said fusion proteins, and the assembly of said fusion proteins to form chimeric VLPs.

A preferred embodiment of this second aspect of the invention relates to a process for obtaining the chimeric VLP particles of the invention, which comprises culturing a host cell comprising a nucleic acid encoding the fusion protein of the invention, under conditions that allow the expression of said fusion proteins, and the assembly of said fusion proteins to form chimeric VLPs, and which further comprises isolating or purifying said chimeric VLPs.

The fusion protein of the invention can be obtained by genetic or recombinant engineering techniques well known in the state of the art. The sequence of a nucleic acid encoding the fusion protein of the invention (hereinafter, nucleic acid of the invention) can be obtained by any biological or synthetic method, including, for example, but not limited to, the restriction of appropriate sequences or amplification of the DNA sequence of the protein of interest by polymerase chain reaction (PCR).

In a preferred embodiment, the nucleic acid of the invention comprises the sequence SEQ ID NO: 5.

The nucleic acid may be comprised in a gene construct (hereinafter, gene construct of the invention). This gene construct of the invention may comprise the nucleic acid of the invention, operably linked to, a sequence regulating the expression of the nucleic acid of the invention, thereby constituting an expression cassette. "Operationally linked" refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way. A control sequence "operatively linked" to the nucleic acid is linked to it in such a way that expression of the nucleic acid coding sequence is achieved.

"Control sequence" refers to nucleic acid sequences that affect the expression of the sequences to which they are linked. Such control sequences include, for example, but not limited to, promoters, initiation signals, termination signals, enhancers or silencers. The term "control sequences" is intended to include those components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.

In a preferred embodiment, the gene construct of the invention comprises the nucleic acid of the invention operably linked to at least one control sequence of the list comprising: a. a promoter,

b. a transcription initiation signal,

C. a transcription termination signal,

d. a polyadenylation signal, or

and. a transcriptional activator.

As used herein, the term "promoter" refers to a region of DNA located at position 5 'with respect to the starting point of transcription and which is necessary or facilitates such transcription in an animal cell. This term includes, for example, but not limited to, constitutive promoters, cell or tissue specific promoters or inducible or repressible promoters.

The control sequences depend on the origin of the cell in which the nucleic acid of the invention is to be expressed. In a particular embodiment, the expression control sequences linked to the nucleic acid of the invention are functional in prokaryotic cells and organisms, for example, but without limit yourself, bacteria; while in another particular embodiment, said expression control sequences are functional in eukaryotic cells and organisms, for example, yeast cells or animal cells. The nucleic acid of the invention or the gene construct of the invention can be introduced into a cell, called a host cell, for example, but not limited, as a naked nucleic acid or by a vector. The term "cloning vector", as used in the present description, refers to a DNA molecule in which another DNA fragment can be integrated, without losing the capacity for self-replication. Examples of expression vectors are, but are not limited to, plasmids, cosmids, DNA phages or artificial yeast chromosomes.

The term "expression vector", as used herein, refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a cell, called a host cell. Said nucleic acid is generally operatively linked to control sequences.

The term "host cell", as used herein, refers to any prokaryotic or eukaryotic organism that is the recipient of an expression vector, cloning or any other DNA molecule.

A third aspect of the invention relates to the use of the chimeric VLP of the invention for the preparation of a medicament, preferably a vaccine. A fourth aspect of the invention relates to the use of the chimeric VLP of the invention for the preparation of a medicament for the prevention and / or treatment of an infection caused by the influenza virus. A sixth aspect of the invention relates to a pharmaceutical composition (hereinafter, pharmaceutical composition of the invention) comprising the chimeric VLP of the invention. A preferred embodiment of this sixth aspect of the invention relates to a pharmaceutical composition comprising the chimeric VLP of the invention and further comprising a pharmaceutically acceptable carrier. Another preferred embodiment of this sixth aspect of the invention relates to a pharmaceutical composition comprising the chimeric VLP of the invention and further comprising another active ingredient. A more preferred embodiment of this sixth aspect of the invention relates to a pharmaceutical composition comprising the chimeric VLP of the invention, a pharmaceutically acceptable carrier and also another active ingredient. As used herein, the terms "active substance", "active substance", pharmaceutically active substance "," active ingredient "or" pharmaceutically active ingredient "refers to any component that potentially provides a pharmacological activity or other different diagnostic effect , cure, mitigation, treatment or prevention of a disease, or that affects the structure or function of the body of the human being or other animals.

The pharmaceutical composition of the invention can be formulated for administration in a variety of ways known in the state of the art. Such formulations may be administered to an animal and, more preferably, to a mammal, including a human, by a variety of routes, including, but not limited to parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal, intraaricular, intrasynovial. , intrathecal, intralesional, intraarterial, intracapsular, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular or topical. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, age, weight, sex or tolerance of the animal. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the pharmaceutically effective composition that produces the desired effect and, in general, will be determined among other causes, by the characteristics of said pharmaceutical composition and of the therapeutic effect to be achieved. The pharmaceutically acceptable "adjuvants" or "vehicles" that can be used in said compositions are the vehicles known in the state of the art.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES

Figure 1: Construction of insertion vectors: Represents the insertion of Notl-Spel restriction sites from Serine-Threonine (TS) insertions generated by directed mutagenesis. In Step 1 an adapter is cloned by using Xbal-Spel compatible restriction enzymes. In Step 2 the adapter is replaced by the insert of interest using the Notl-Spel restriction sites.

Figure 2: DNA sequence of the 2xM2e insert and its corresponding amino acid sequence. VRN codons (V = nucleotides A, C or G; R = nucleotides A or G; N = nucleotides T, C, A or G) encode the 9 most hydrophilic amino acids (h), which are histidine (H), glutamine ( Q), asparagine (N), lysine (K), aspartic acid (D), glutamic acid (E), arginine (R), serine (S) and glycine (G). The sequence of M2e is represented with gray color.

Figure 3. VP2-2xM2e chimeric VLP. Electron microscopy of birnavirus chimeric pseudoviral capsids (VLPs) that have the M2e influenza virus antigen incorporated into the external Hl domain of IBDV VP2 ₄₅ 2 protein.

EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1: SEARCH AND SELECTION OF VIRAL CAPSIDES OF BIRNAVIRUS CONTAINING THE M2e ANTIGEN OF THE VIRUS OF THE FLU. a) SEARCH AND SELECTION, THROUGH A PROCESS OF SUCCESSIVE SCREENING, OF CHEMICAL VLP CONTAINING M2e OF THE VIRUS OF THE FLU.

For the search of places of insertion of the M2e antigen that result in the effective formation of chimeric VLPs, a process of insertion of two copies of M2e of the influenza virus is carried out in different positions of the external P domains of the VP2 protein. For this, a collection of 8 different plasmids containing a Spel site at the preferred positions within the domains is obtained by directed mutagenesis using the yeast expression plasmid pESC-URA (Stratagene ™) -VP2 ₄₅ 2 external P of the VP2 protein. To limit the number of false positives during searches, a second generation of insertion vectors in the external P-domains of the VP2 protein is prepared so that each plasmid contains a multiple cloning site (MCS). the directional cloning of the DNA of interest. As shown in Figure 1, for this purpose plasmids containing the Spel site at each of the chosen insertion points are digested with the Spel restriction enzyme and ligated to incorporate the insert containing an MCS with the Notl and Spel restriction sites, which allow the subsequent directional cloning of inserts of interest. The insert with the MCS contains several stop codons of translation "stop" in phase, which ensures that the religion of vectors without insert generates a truncated VP2, unable to form VLP. The selection of the 8 vectors includes cloning sites in the 4 main external domains of the P region of the VP2 protein (see Table 1).

Table 1

For cloning and subsequent identification of the 2xM2e inserts that give rise to chimeric VLPs, the 2xM2e sequence containing two copies of the sequence encoding the human influenza virus M2e antigen is generated by PCR (SEQ ID NO: 3) in which methionine in position 1 (Mi) is excluded, and the cysteines of positions 17 and 19 (Ci ₇ and C19) are replaced by serines (S) (SEQ ID NO: 4).

In the design and construction of the insert the two copies of M2e are separated from each other and flanked with respect to the VP2 sequences by a "linker" consisting of four hydrophilic amino acids (h) "hhhh- (M2e) -hhhh- (M2e) -hhhh "[SEQ ID NO: 5]. In the molecular design for cloning, each of the four hydrophilic amino acids is encoded by a codon of three randomly generated nucleotides as follows: in position 1 nucleotide A, G or C (V according to the IUPAC code), in the position 2 nucleotide A or G (R according to the IUPAC code), and in position 3 nucleotide A, G, C or T (N according to the IUPAC code). The resulting VRN codons code for the 9 most hydrophilic amino acids, that is, histidine (H), glutamine (Q), asparagine (N), lysine (K), aspartic acid (D), glutamic acid (E), arginine (R ), serine (S) and glycine (G), as shown in Figure 2. In addition to these "hydrophilic amino acid linkers" (h), the different randomly generated 2xM2e inserts contain seven additional amino acids in its ends whose DNA sequence contains the Not \ and Spel restriction sites, used in molecular cloning [SEQ ID NO: 6]. The generation of an insertion library "hhhh- (M2e) -hhhh- (M2e) -hhhh" in the eight possible preselected insertion sites in the P region of the VP2 protein is carried out by a ligation reaction of the library of 8 vectors pre-digested with Not \ and Spel and the library of PCR-generated DNA fragments containing the randomly generated 2xM2e fragments and pre-digested with Not \ and Spel. The ligation result is transformed into electrocompetent E.coli cells to generate a library of clones containing the different 2xM2e fragments inserted in the eight possible points of Preset insert in VP2. After library expansion, ^ g of DNA from a mixture of plasmid DNA from all clones of the library is used to transform yeast cells, S.cerevisiae strain Y449 that are subsequently seeded in YNB / CSM-URA medium with 2% glucose The yeast clones isolated and obtained in this selective medium will be transferred to YNB / CSM-URA plates with galactose and the resulting colonies are transferred to polyvinylidene fluoride membranes (English PVDF, Polyvinylidene Fluoride) to study the production of VLP by immunoblots of colonies using specific antibodies against the M2e epitope. A selection of 50 positive clones are replicated and cultured individually in YNB / CSM-URA selective liquid medium with 2% galactose and quantitative sandwich ELISA assays are performed with the extracts resulting from their lysis to confirm and the production of VLP by using specific antibodies against IBDV VLP obtained from mouse and rabbit. Eleven colonies with VLP formation efficiencies above 10% are sequenced to identify the 2xM2e insert and its insertion site within the VP2 protein. The confirmed positive clones are shown in Table 2. Table 2: Insertion sites in VP2 and insert sequence of 2xM2e in the constructions resulting from the process of search and selection of chimeric VLPs containing 2xM2e and expressing more efficiently.

Clone Construction Site%

INSERT SEQUENCE

resulting erción

ID. VLP Ins

G ₂₅₄ TTRGGRGGGDSLLTEVETPIRN

1A G254 † L255 G ₂ 54 † 2xM2e † L ₂ 55 19.5 EWGSRSNDSSDQQGHSLLTEVETP

IRNEWGSRSNDSSDQKQKTS † L ₂ 55

G254TTRGGRRENQSLLTEVETPIRN

1 B G254 † L255 G ₂ 54 † 2xM2e † L ₂ 55 15.2 EWGSRSNDSSDRREQSLLTEVETP

IRNEWGSRSNDSSDGEDKTS † L ₂ 55

2A T286 † D287 T ₂ 86 † 2xM2e † D ₂ 87 15.9 T ₂₈ 6TRIGGRGDGGSLLTEVETPIRN

EWGSRSNDSSDKNNHSLLTEVETP IRNEWGSRSNDSSDRKNTS † D ₂₈₇

T ₂₈ 6 † TRGGRGGGRSLLTEVETPIRN

2B 286 † D287 T ₂ 86 † 2xM2e † D ₂ 87 12.4 EWGSRSNDSSDRENRSLLTEVETP

IRNEWGSRSNDSSDRRRNTS † D ₂₈ 7

T ₂₈ 6 † TRGGREDGGSLLTEVETPIRN

2C T286 † D287 T ₂ 86 † 2xM2e † D ₂ 87 18.4 EWGSRSNDSSDKGEGSLLTEVETP

IRNEWGSRSNDSSDRRHETS † D ₂₈₇

Q ₃₂ or † TRGGRSHSRSLLTEVETPIRN

3A Q320 † A321 Q ₃ 2nd † 2xM2e † A ₃ 2i 31, 6 EWGSRSNDSSDRQKKSLLTEVETP

IRNEWGSRSNDSSDKRDQTS † A ₃₂₁

Q ₃₂ or † TRGGRDRGDSLLTEVETPIRN

3B Q320 † A321 Q ₃ 2nd † 2xM2e † A ₃ 2i 31, 1 EWGSRSNDSSDSQNESLLTEVETP

IRNEWGSRSNDSSDGHGRTS † A ₃₂₁

D ₃₂₃ † TRGGRQREQSLLTEVETPIRN

4A D323 † Q324 D ₃₂₃ † 2xM2e † Q ₃₂ 4 35.2 EWGSRSNDSSDQQGESLLTEVETP

IRNEWGSRSNDSSDGHGRTS † Q ₃₂₄

Continued Table 2:

b) ANSWER ANSWERS OF DIFFERENT VLP INCORPORATING M2e.

The identification of chimeric VLP VLP-M2e capable of generating a significant immune response against M2e is carried out by immunizing 6 mice with an initial dose intraperitoneally (i. P.), Followed by two recall doses subcutaneously. (sc) - In each dose 50 μg is administered for each of the VLP-2xM2e generated, or of the VLPs control without the M2e insert, in 200 μΙ saline. All three doses are administered at intervals of 3 weeks. One week before the first immunization and two weeks after each booster dose, blood samples are obtained from the animals by retro-orbital puncture. The samples are treated at 37 ^° C for 60 minutes, followed by a brief incubation on ice and then, by two successive centrifugations, recover the serum from the supernatant in both centrifuges. Serum samples are stored at -20 ° C. Antibody titers are determined by ELISA tests using mixtures of sera from animals of the same group. For titration of specific anti-M2e antibodies, vinyl microplates (Corning ™, Costar Ref. 2595) are upholstered with 50 μΙ per well of a solution of the M2e peptide at 2μg / ml in 50mM sodium bicarbonate buffer, pH 9.7, and incubate overnight at 4 ° C. After washing, the plates are blocked for 1 hour with TBS, 0.05% Tween-20, 3% BSA, and after that serial dilutions 1/3 of the different serum samples are added, starting with a 1/100 dilution. Bound antibodies are detected by adding antibodies conjugated to total anti-lgG peroxidase (Jackson ImmunoResearch ™, Code 1 15-035-003) followed by a 5 min incubation with the peroxidase substrate (SIGMAF ¾ST ™ OPD, ref. P9187, Sigma-Aldrich ™). After stopping the reaction with 20 μΙ of 3M HCI, the absorbance value is measured at a wavelength of 495 nm. The "endpoint" titers are defined as the highest dilution that produces an optical density (OD) value twice the value of the negative control (preimmune serum).

Table 3: Places of insertion in VP2 and sequence of the 2xM2e insert of the constructions resulting from the process of search and selection of chimeric VLPs containing 2xM2e and expressing more efficiently.

Clone Construction Site Anti IgG

% VLP

Resulting insertion

ID. "endpoint"

1A G254ÍI-255 G ₂ 54 † 2xM2e † L ₂ 55 19.5 1: 14,000

1 B G254 † l-255 G ₂ 54 † 2xM2e † L ₂ 55 15.2 ND

2A 286 † D287 T ₂ 86 † 2xM2e † D ₂ 87 15.9 1: 8,500

2B T286 † D287 T ₂ 86 † 2xM2e † D ₂ 87 12.4 ND

2C 286 † D287 T ₂ 86 † 2xM2e † D ₂ 87 18.4 1: 1 1,000

3A Q-320 † A321 Q ₃ 2nd † 2xM2e † A ₃ 2i 31, 6 1: 22,000

3B Q-320 † A321 Q ₃ 2nd † 2xM2e † A ₃ 2i 31, 1 ND

4A D323 † Q-324 D ₃₂₃ † 2xM2e † Q ₃₂ 4 35.2 1: 35,000

4B D323 † Q-324 D ₃₂₃ † 2xM2e † Q ₃₂ 4 24.5 1: 28,000

4C D323 † Q-324 D ₃₂₃ † 2xM2e † Q ₃₂ 4 24.4 1: 21,000

4D D323 † Q324 D ₃₂₃ † 2xM2e † Q ₃₂ 4 34.8 1: 28,000

C - VLP control 100 <1: 500

c) BIOLOGICAL EFFECTIVENESS OF VLP INCORPORATING M2e.

The efficacy as a vaccine for the protection against influenza virus infection of the three candidates of VLP-M2e with greater title "endpoint" is evaluated by the viral challenge in previously immunized animals. For this, groups of 13 mice are immunized with an initial immunogen dose intraperitoneally (i.p.), followed by two subcutaneous (s.c.) recall doses at 3-week intervals. In each dose 50 μg of VLP-2xM2e, or of the appropriate control, is administered in 200 μΙ of saline.

Three weeks after the second recall dose (day 65), the animals are exposed to a dose of 4xLD ₅ or (4 times the 50% lethal dose) of a strain of the mouse-adapted Influenza A virus (A / Puerto Rico8 / 34, H1 N1) and mortality and clinical symptoms are recorded for 17 days. According to the rules of ethics of experimentation in animals, mice are sacrificed if their body weight falls below 75% of their initial weight. As shown in Table 4, all mice in the control VLP groups without M2e inserts die within 8 days after the viral challenge. In contrast, none of the animals vaccinated with VLP ₄ 52-D ₃ 23 † 2xM2e † Q324 (Clone ID 4A) die in the course of the experiment, which means a survival rate of 100%.

Table 4: Survival data of mice vaccinated with different VLPs incorporating 2xM2e insertions in P regions of the VP2 of IBDV.

Clone Construction Site Anti IgG Challenge

% VLP

ID. Insertion resulting "viral endpoint"

1A G254 † L255 G ₂ 54 † 2xM2e † L ₂ 55 19.5 1: 14,000 55%

2C T286 † D287 T ₂ 86 † 2xM2e † D ₂ 87 18.4 1: 1 1 .000 45%

4A D323 † Q324 D ₃₂ 3 † 2xM2e † Q ₃ 24 35.2 1: 35,000 100%

C - VLP control 100 <1: 500 0%

Claims

1. Chimeric pseudo-viral particle (VLP) formed by a fusion protein comprising:

- a subunit (b) comprising an influenza virus M2e antigen,

where the subunit (b) is inserted in the subunit (a).

2. Chimeric pseudo-viral particle according to claim 1, wherein the subunit (a) consists of a protein with at least 80% identity with SEQ ID NO: 1 or a fragment thereof.

3. Chimeric pseudo-viral particle according to claim 2, wherein the subunit (a) consists of a protein with SEQ ID NO: 1 or a fragment thereof.

4. Chimeric pseudo-viral particle according to claim 3, wherein the subunit (a) is SEQ ID NO: 2.

5. Chimeric pseudo-viral particle according to any one of claims 1 to 4, wherein the subunit (b) is inserted into the P region of the subunit (a).

6. The chimeric pseudo-viral particle according to claim 5, wherein the subunit (b) is inserted into the Hl domain of the P region of the subunit (a).

7. The chimeric pseudo-viral particle according to claim 6, wherein the subunit (b) is inserted between amino acids D323 and Q324 of the Hl domain of the P region of the subunit (a).

8. Chimeric pseudo-viral particle according to claim 5, wherein the subunit (b) is inserted into the DE domain of the P region of the subunit

(to) .

9. Chimeric pseudo-viral particle according to any one of claims 1 to 8, further comprising one or two hydrophilic spacer polypeptides between the amino acid sequence of the subunit

(b) and the amino acid sequences of the subunit (a).

10. Chimeric pseudo-viral particle according to any one of claims 1 to 9, wherein the subunit (b) comprises at least two M2e antigens.

eleven . Chimeric pseudo-viral particle according to claim 10, wherein the subunit (b) comprises two M2e antigens.

12. Chimeric pseudo-viral particle according to any of claims 10 or 1, wherein the subunit (b) further comprises at least one hydrophilic spacer polypeptide between the two M2e antigens.

13. Chimeric pseudo-viral particle according to any of claims 1 to 12, wherein the M2e antigen is a polypeptide with at least 60% identity with SEQ ID NO: 3.

14. Chimeric pseudo-viral particle according to any of claims 1 to 13, wherein the M2e antigen is a polypeptide with the amino acid sequence SEQ ID NO: 4.

15. Chimeric pseudo-viral particle according to claim 1, wherein a polypeptide with the amino acid sequence SEQ ID NO: 7 is inserted into SEQ ID NO: 2.

16. Chimeric pseudo-viral particle according to claim 1, whose amino acid sequence is SEQ ID NO: 8.

17. Method for obtaining chimeric pseudo-viral particles according to any one of claims 1 to 16, comprising culturing a host cell comprising a nucleic acid encoding a fusion protein according to any of claims 1 to 16 under conditions that they allow the expression of said fusion proteins, and the assembly of said fusion proteins to form chimeric pseudo-viral particles.

18. Method for obtaining chimeric pseudo-viral particles according to claim 17 wherein the nucleic acid comprises the sequence SEQ ID NO: 5.

19. Method for obtaining chimeric pseudo-viral particles according to any of claims 17 or 18, and further comprising isolating or purifying said chimeric pseudo-viral particles.

20. Use of the chimeric pseudo-viral particle according to any of claims 1 to 16 for the preparation of a medicament.

twenty-one . Use of the pseudo-viral particle according to any of claims 1 to 16 for the preparation of a medicament for the prevention and / or treatment of an infection caused by the influenza virus.

22. Pharmaceutical composition comprising the chimeric pseudo-viral particle according to any one of claims 1 to 16.

23. Pharmaceutical composition according to claim 22 further comprising a pharmaceutically acceptable carrier.

24. Pharmaceutical composition according to any of claims 22 or 23 which further comprises another active ingredient.